Specific aims: 1) To investigate whether the reactivation of glucagon-inhibited pyruvate kinase in rat hepatocytes by insulin is due to a decrease in the phosphorylation of the enzyme. 2) To investigate in collaboration with prof. Leonard Jarett, Philadelphia, whether insulin mediator described by his and prof. J. Larner's groups reactivates pyruvate kinase when added to intact or broken glucagon-treated hepatocytes. 3) To study the mechanism of action of the mediator, especially effects on cAMP-dependent protein kinase and phosphoprotein phosphatases with pyruvate kinase as substrate. 4) To study the effects of the mediator on other phosphorylatable liver enzymes, e.g. FDPase, phenylalanine hydroxylase and ATP-citrate lyase. 5) To look for an insulin-stimulated protein kinase in hepatocytes and adipocytes which should be active on ATP-citrate lyase and ribosomal protein S6. Methodology: Hepatocytes are isolated after collagenase treatment and incubated with glucagon which inhibits pyruvate kinase at a low phosphoenolpyruvate concentration due to a cAMP-stimulated phosphorylation. Reactivation of pyruvate kinase will be studied after addition of the insulin mediator (obtained from prof. Jarett) or insulin to intact and broken cells. The activity changes will be correlated to the degree of phosphorylation by isolating 31P-labeled enzyme from 32Pj-incubated cells using immunochemical methods. Corresponding studies will be done with other phosphorylatable enzymes (FDPase phenylalanine hydroxylase, ATP-citrate lyase) to investigate the universality of the mediator. Long-term objectives: For a long time very little has been known about the mechanism of action of insulin on a molecular level. The finding of the mediator by Larner's and Jarett's groups seems to be the most promising contribution within the field. Liver pyruvate kinase appears to be a very suitable enzyme for studies of insulin-induced dephosphorylation, since the enzyme only has one phosphate-accepting site. Our group has a unique experience of this system which should be a good complement to the intramitochondrial pyruvate dehydrogenase system.